The epithelial sodium channel (ENaC) plays an important role in maintaining sodium balance, blood volume, and blood pressure. It is localized to the apical membrane of various epithelial cells including those that line the distal renal nephron. Prior observations suggest that the actin cytoskeleton plays a role in the regulation of ENaC activity and it is known that ENaC is regulated by phosphoinositides (i.e. PIP2 and PIP3). However, the mechanism by which the actin cytoskeleton regulates ENaC and how these rare phosphoinositides are presented to ENaC is unknown. This proposal describes experiments to investigate the hypothesis that the actin cytoskeleton serves as an organizing center for MARCKS, calmodulin, and ENaC and that this organization is necessary for MARCKS-dependent regulation of ENaC activity by phosphoinositides. We will use various molecular and proteomic techniques (e.g., co-immunoprecipitation studies, GST pull-down assays, and fluorescence resonance energy transfer (FRET)) to identify cytoskeletal-associated proteins involved in the phosphoinositide-dependent regulation of ENaC and to determine which ENaC domains differentially bind phosphoinositides. We will use electrophysiological methods with specific pharmacological agents to determine the role of MARCKS and phosphoinositides in regulating apical membrane ENaC activity in Xenopus distal nephron epithelial cells expressing endogenous ENaC. We will perform Fluorescence Recovery after Photobleaching (FRAP) to determine the role of the actin cytoskeleton in the formation of a membrane signaling complex of MARCKS, calmodulin, and ENaC. The overall goal of this investigation is to understand the mechanism by which ENaC is regulated by the actin cytoskeleton, MARCKS, and phosphoinositides and to gain a better understanding for the control of ENaC in health and disease.